Introduction
With the growing understanding of surface receptor structure, signal transduction and function of second messenger systems in eukaryotic cells a new realm of regulatory mechanisms have attracted the interest of many scientists. Altering cell function by intervening with the intracellular controlling systems appears to be a powerful and highly efficient way to subdue unwarranted reactions to environmental factors or microbial infections. In immunology it has become increasingly important to control inflammatory reactions in autoimmunity and allergy, to enhance anti-tumor immunity as well as to increase efficiency in vaccine take. Therefore, exploring the possibilities of immunomodulation through signal-transduction and second messengers may open up new therapies and strategies to treat diseases in which the immune system is part of the pathogenic mechanism or important for host resistance against infectious diseases or development of cancer. Also, therapies preventing organ transplant rejection are highly desired and recent findings in experimental animal models have given evidence that immunomodulation may significantly prolong organ transplant survival time.
There is a group of bacterial toxins that exert strong enzymatic activity on mammalian cells. These toxins, of which E coli heat-labile toxin (LT) and cholera toxin (CT) are well known representatives, act by ADP-ribosylation of GTP-binding proteins in the cell membrane of the target cells. In the ADP-ribosylation reaction, NAD+ is split into free nicotinamide and an ADP-ribose moiety is associated with the guanidinium group of an arginine in the a-subunit of the stimulatory G protein (Gs). The Gsa-protein becomes permanently activated and stimulates adenylate cyclase which results in the formation of large quantities of intracellular cAMP. The increase in cAMP may then act to immunomodulate many diverse immune reactions such as increasing B lymphocyte differentiation, augmenting co-stimulation of antigen-presenting cells, inhibiting or promoting various T cell functions or modulating apoptosis in lymphoid cells. There is no more important and ubiquitos intraellular regulatory molecule than cAMP. The mechanism by which cAMP exerts its function is pleiotropic and increases in intracellular cAMP may affect different cells in different ways. Also, cells of a particular cell lineage may respond with inhibited function to cAMP at one stage of differentiation whereas at a different stage strong enhancing effects may be observed. Many genes have been found to be under transcriptional control of cAMP, and cAMP regulatory gene elements have been described.
CT is composed of five enzymatically inactive, non-toxic B-subunits (CTB) held together in a pentamere structure surrounding a single A-subunit that contains a linker to the pentamere via the A2 fragment (CTA2) and the toxic enzymatically active A1-fragment (CTA1) of the molecule. The toxic CTA1 has strong ADP-ribosyl transferase activity and is thought to act on several G-proteins of which the activity is strongest on Gsa. This results in activation of adenylate cyclase and the subsequent intracellular increase in cAMP. CTB binds to the ganglioside GMl-receptor, present on most mammalian cells including lymphocytes and gut epithelial cells, and CTA is thereafter translocated into the cell-membrane/cytosol of the cell where the CTA1 and CTA2 are dissociated. The profuse diarrheal response in cholera is thought to result from CTA1-induced increased cAMP levels in the intestinal epithelium. An important issue is, therefore, whether the immunomodulating property of CT may be separated from the toxic property.
Exploitation of the mucosal immune system offers several advantages from a vaccine point of view. Mucosal vaccines may achieve both systemic and local mucosal immune protection against infectious microorganisms of which many gain access to the body via mucosal membranes There is a growing interest for oral vaccines and for the possibility of using such vaccines to protect against infectious diseases affecting not only mucosal surfaces but also against diseases like HIV, polio etc Holmgren, J. and Lycke, N. In 11th Nobel Conference; Chartwell-Bratt Ltd, Bromley: 1986; pp 9-22.; Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990.!. However, most soluble protein antigens are poorly immunogenic when given alone perorally and, although live vectors have been found to be efficient delivery systems for oral antigen administration, the general use of such vaccines is still unclear Holmgren, J. and Lycke, N. In 11th Nobel Conference; Chartwell-Bratt Ltd, Bromley: 1986; pp 9-22; Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990.! This has prompted research with the aim of identifying mucosal adjuvants that could find general use in non-replicating oral vaccines. A powerful mucosal adjuvant should be non-toxic and greatly improve immunogenicity and generate immunological memory to soluble antigens which are normally poor immunogens when administered perorally Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990.!. Two principally different approaches have been taken to achieve this goal; the first has focused on constructing powerful delivery systems for oral antigens such as encapsulated microparticles or immune stimulating complexes (iscomes) Challacombe, S. J., Rahman, D., Jeffery, H., Davis, S. S. and O'Hagan, D. T. Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen. Immunology. 76, 164-8, 1992.; Eldridge, J., Gilley, R. M., Staas, J. R., Moldevanue, Z., Meulbroek, T. and Tice, T. R. Biodegradable microspheres: vaccine delivery system for oral immunization. In Current topics in Microbiology and Immunology. 1989; Vol. 146; pp 59.!. The second approach is to construct an adjuvant that will modulate and greatly augment the immune response to the oral antigen by evoking strong IgA immunity and immunological memory in the gut mucosa as well as at other mucosal sites Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990.!.
Cholera toxin is an exceptionally potent mucosal immunogen and adjuvant Elson, C. 0. and Ealding, W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol. 132, 2736-41, 1984.; Liang, X. P., Lamm, M. E. and Nedrud, J. G. Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus. J Immunol. 141, 1495-501, 1988.; Lycke, N. and Holmgren, J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology. 59, 301-8, 1986!. The toxin has become the best studied prototype and most used probe to understand how an efficient mucosal adjuvant might be constructed Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990!. Despite several years of research and many reports on immunomodulating properties of CT it is still unclear which entity of CT that is critical for its adjuvant mechanism. The adjuvant properties of CT have been studied in a mouse model using both in vivo and in vitro experimental systems Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990; Lycke, N. and Holmgren, J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology. 59, 301-8, 1986; Bromander, A., Holmgren, J. and Lycke, N. Cholera toxin stimulates IL-1 production and enhances antigen presentation by macrophages in vitro. J Immunol. 146, 2908-14, 1991.; Hornqvist, E., Goldschmidt, T. J., Holmdahl, R. and Lycke, N. Host defense against cholera toxin is strongly CD4+ T cell dependent. Infect Immun. 59, 3630-8, 1991; Lycke, N. and Holmgren, J. Long-term cholera antitoxin memory in the gut can be triggered to antibody formation associated with protection within hours of an oral challenge immunization. Scand J Immunol. 25, 407-12, 1987.; Lycke, N. and Strober, W. Cholera toxin promotes B cell isotype differentiation. J Immunol. 142, 3781-7, 1989.; Lycke, N. and Holmgren, J. Adoptive transfer of gut mucosal antitoxin memory by isolated B cells 1 year after oral immunization with cholera toxin. Infect Immun. 57, 1137-41, 1989; Lycke, N., Bromander, A. K., Ekman, L., Karlsson, U. and Holmgren, J. Cellular basis of immuno-modulation by cholera toxin in vitro with possible association to the adjuvant function in vivo. J Immunol. 142, 20-7, 1989; Lycke, N., Karlsson, U., Sjolander, A. and Magnusson, K. E. The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens. Scand J Immunol. 33, 691-8, 1991.; Lycke, N., Severinson, E. and Strober, W. Molecular effects of cholera toxin on isotype differentiation. Immunol Res. 10, 407-12, 1991.; Lycke, N., Tsuji, T. and Holmgren, J. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur J Immunol. 22, 2277-81, 1992.; Lycke, N. Y. Cholera toxin promotes B cell isotype switching by two different mechanisms. cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GMl-receptor binding enhances later events in differentiation. J Immunol. 150, 4810-21, 1993!. In the research we have tried to systematically address CT's immunomodulating properties on the different events involved in initiating and regulating mucosal immune responses; antigen-presentation, IgA B cell differentiation, T cell regulation and development of long-term immunological memory Lycke, N. and Svennerholm, A. -M. The presentation of immunogens at the gut and other mucosal surfaces. In The molecular approach to new and improved vaccines.; Woodrow and Levine, Ed.; Marcel Dekker Inc: NY, 1990.; Lycke, N. and Holmgren, J. Adoptive transfer of gut mucosal antitoxin memory by isolated B cells 1 year after oral immunization with cholera toxin. Infect Immun. 57, 1137-41, 1989; Lycke, N., Severinson, E. and Strober, W. Molecular effects of cholera toxin on isotype differentiation. Immunol Res. 10, 407-12, 1991.; Vajdy, M. and Lycke, N. Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant. Immunology. 80, 197-203, 1993!. Previous data and the finding of an increased gut permeability for luminal antigens in the presence of CT suggested that the adjuvant effect might depend on the ability of CT, but not CTB, to activate the adenylate cyclase/cAMP system. Liang and co-workers Liang, X. P., Lamm, M. E. and Nedrud, J. G. Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus. J Immunol. 141, 1495-501, 1988! have shown that glutaraldehyde treatment of CT leads to a 1000-fold reduction in toxicity but preserved capacity to enhance mucosal immune responses after oral immunization. A concomitant loss of ribosyltransferase activity occurred such that with a suitable substrate and necessary co-factors (supplied by lysed red cells) 5-10% residual cyclic AMP was generated compared to untreated toxin.